The mRNA cap added to the 5’ end of nascent transcripts is required for the efficient gene expression in eukaryotes. In vertebrates, the guanosine cap is methylated at N7 position by RNMT, which is in complex with its activating subunit RAM. Additionally, the first and second transcribed nucleotides can be methylated at ribose O2 position by CMTR1 and CMTR2 respectively. The mRNA cap protects transcripts from degradation and recruits cap-binding factors to promote pre-mRNA processing, nuclear export and translation initiation. In mouse embryonic stem cells (mESCs), high levels of RAM maintain expression of pluripotency factors. Differentiation of mESCs to neural progenitors is accompanied by a suppression of RAM, resulting in downregulation of pluripotency factors and efficient formation of neural cells. Here, I demonstrated that the suppression of RAM during neural differentiation is promoted via ubiquitination and proteasomal degradation. Concurrently, neural differentiation is associated with an increase in CMTR1 expression, creating a developmental cap methyltransferase switch. Moreover, differentiation into endodermal and mesodermal lineages exhibited distinct changes in the mRNA capping enzymes expression. In mESCs, RAM promotes expression of translation-associated proteins and promotes global loading of mRNA on ribosomes. RAM contributes to the ESC-specific gene expression program, by maintaining optimal expression of pluripotency-associated transcripts and inhibiting expression of neural genes. Chromatin immunoprecipitation revealed that RAM, RNMT and CMTR1 promote binding of RNA polymerase II at gene loci. In RAM-repressed cells, RNA polymerase II binding was reduced at pluripotency-associated genes, but relatively increased at neural genes. Moreover, knock-down of RNMT or CMTR1 induced increased or decreased accumulation of RNA polymerase II at promoter proximal regions respectively. In naïve T cells, Rnmt or Cmtr1 conditional knock-outs caused downregulation of translation-related transcripts and upregulation of cell cycle transcripts. Furthermore, many transcripts were specifically dependent on RNMT or CMTR1 for expression, demonstrating distinct roles of these cap methyltransferases. Thus, the mRNA cap methylation emerges as an important regulator of pluripotency and differentiation, modulating gene expression at transcriptional and post-transcriptional levels.

L’ARN polymérase II (ARNPII) synthétise des ARNs codants et des ARNs non-codants (ARNnc) tels que les petits ARNs nucléaire/nucléolaire (sn/snoRNAs) et les CUTs (Cryptic Unstable Transcripts). Les CUTs sont des transcrits ubiquitaire souvent produits dans des régions codants dont la transcription peut interférer avec l’expression des gènes. Le contrôle de l’expression des ARNnc est essentiel et se fait aux niveaux de la terminaison de la transcription et la dégradation de l’ARN. Chez la levure Saccharomyces cerevisiae la terminaison de la transcription des gènes codants est effectuée par le Facteur de Clivage et de Polyadénylation (CPF), tandis que les ARNnc courts sont terminés par le complexe Nrd1p-Nab3p-Sen1p (NNS). La terminaison de la transcription est régulée par la phosphorylation du domaine C-terminal (CTD) de l’ARNPII composé de répétitions du motif Y1S2P3T4S5P6S7. Un niveau élevé de phosphorylation des résidus Ser5 près du promoteur permet l’activité du complexe NNS. La phosphorylation des résidus Ser2 est catalysée durant la transcription par la kinase Ctk1p et ces résidus sont reconnus par des éléments de la voie CPF. Mon travail de thèse a porté sur le mécanisme de terminaison de la transcription par le complexe NNS. La terminaison NNS dépend de la liaison de Nrd1p et Nab3p à des motifs dans l’ARN naissant et l’activité hélicase de Sen1p qui provoque le relarguage de la polymérase. La sous-unité Nrd1p interagit avec le domaine CTD de l’ARNPII phosphorylé sur Ser5 à travers son domaine CID (CTD-interaction domain). Le rôle du CID dans la terminaison à été proposé mais pas encore clairement démontré. En collaboration avec le groupe de P. Cramer (Université Louis-et-Maximilien de Munich Allemagne) nous avons mis en évidence que le CID est requis pour une terminaison efficace par la voie NNS et qu’il est important pour le recrutement de Nrd1p sur l’ARNPII. Le CID est aussi impliqué de manière directe ou indirecte dans l’interaction de Sen1p avec Nrd1p et Nab3p. En parallèle, avec le groupe de F. Holstege (Université Centre Médicale de Utrecht, Pays-Bas) nous avons montré que la phosphorylation en Ser2 du domaine CTD est requise pour une terminaison efficace par la voie NNS. De manière surprenante, ce résidu joue un rôle mineur dans la terminaison des ARNs codants effectuée par le complexe CPF. Les ARNs naissant terminés par le complexe NNS sont rapidement ciblés par le complexe nucléase exosome/Rrp6p et son cofacteur TRAMP ce qui mène a la maturation des sn/snoRNAs et la destruction des CUTs. Le complexe NNS interagit in vivo avec l’exosome et le complexe TRAMP, ce qui facilite la dégradation. Cependant les détails moléculaires de cette interaction restent inconnus. Nous avons montré que le domaine CID est requis et suffisant in vivo et in vitro pour l’interaction de Nrd1p avec la partie C-terminale de la sous-unité Trf4p du complexe TRAMP, que nous avons appelé NIM (Nrd1p-Interaction Motif). En collaboration avec le groupe de R. Stefl (Université Masaryk, République Tchèque) nous avons étudié par spectroscopie RMN la structure de ce motif NIM lié au CID. Nous avons mis en évidence que le CID lie le NIM et le CTD de façon similaire, et que ces interactions sont mutuellement exclusives. Le NIM se lie au CID environ 100 fois plus fortement qu’au CTD. Nous proposons que ces interactions alternatives de Nrd1p définissent des formes différentes du complexe NNS, une qui fonctionne dans la terminaison de la transcription, l’autre qui est active dans la dégradation. In vitro l’interaction du NIM avec le CID stimule l’activité poly(A)-polymérase de Trf4p ce qui suggère une fonction importante de cette interaction dans la dégradation. Nous montrons aussi que Rrp6p interagit directement avec Trf4p et cette liaison in vivo sert à recruter le complexe TRAMP à l’exosome Nous proposons que ce jeu serré d’interactions entre les complexes NNS, TRAMP et l’exosome/Rrp6p contribue à augmenter l’efficacité de dégradation de l’ARN in vivo / The RNA polymerase II (RNAPII) synthesizes protein-coding RNAs and many non-coding RNAs (ncRNAs) such as small nuclear/nucleolar (sn-/snoRNAs) and Cryptic Unstable Transcripts (CUTs). CUTs are ubiquitously transcribed including overlapping and antisense to genes, which can interfere with gene expression. Control of ncRNA expression is vital and also operates at the level of transcription termination and RNA degradation.In yeast Saccharomyces cerevisiae transcription of protein-coding genes is terminated by the Cleavage and Polyadenylation Factor (CPF), while short ncRNAs are generated by transcription termination dependent from the Nrd1p-Nab3p-Sen1p (NNS) complex. Transcription termination is regulated by phosphorylation of the carboxy-terminal domain (CTD) of the Rpb1p subunit of RNAPII, composed of repeats of the Y1S2P3T4S5P6S7 motif. Promoter-proximal high levels of serine 5 phosphorylated (Ser5P) CTD favors the function of the NNS pathway while the Ser2 phosphorylated mark (Ser2P), which is gradually introduced during transcription by Ctk1p, is recognized by components of the CPF pathway. The study of the mechanism of action of the NNS complex was the subject of my PhD work.NNS-dependent transcription termination is driven by the recognition of four nucleotide motifs in the nascent RNA by Nrd1p and Nab3p and the release of the RNAPII by the Sen1p helicase. Nrd1p interacts with the CTD-Ser5P via its CTD-interaction domain (CID). Thus a role of the CID in termination was anticipated but not demonstrated. In collaboration with the group of P. Cramer (Ludwig Maximilian University of Munich, Germany), we have shown that the Nrd1p CID domain is required for efficient transcription termination at most NNS-target genes and that it is important for the recruitment of Nrd1p to the RNAPII. This domain is also involved, directly or indirectly, in the interaction of the Sen1p helicase with Nrd1p and Nab3p. In the second project, in collaboration with F. Holstege group (University Medical Center Utrecht, Netherlands), we have shown that the CTD-Ser2P mark is important for efficient transcription termination by the NNS pathway but, surprisingly, it appears to play a minor role in termination of mRNA-coding genes by the CPF-complex.Shortly after NNS-dependent termination, the released ncRNAs are targeted by the nuclear exosome/Rrp6p nuclease complex and its cofactor the TRAMP which results in trimming of sn-/snoRNAs to a mature form and complete degradation of CUTs. The NNS complex co-purifies in vivo with the TRAMP/exosome, which is believed to facilitate subsequent degradation and processing. However, the molecular details of this interaction are unknown. We show that the CID is required and sufficient in vivo and in vitro for the interaction of Nrd1p with a motif present in the C-terminal region of Trf4p, which we called NIM (for Nrd1p-Interaction Motif). In collaboration with the group of R. Stefl (Masaryk University, Czech Republic), we obtained the NMR structure of the CID bound to the NIM and demonstrated that the CID binds in a similar manner to the CTD and the NIM. The CID interacts with the CTD and the NIM in a mutually exclusive manner and the former interaction is roughly 100 times stronger than the first. We propose that these alternative interactions represent two forms of the NNS complex, one functioning in termination and the other in degradation. Importantly, the NIM-CID interaction is likely to be functionally relevant since in vitro it results in the stimulation of the polyA polymerase activity of the Trf4p. We further show that Trf4p interacts directly with Rrp6p, which in vivo serves to recruit the TRAMP to the core exosome complex. This tight interplay between the NNS, TRAMP and exosome/Rrp6p complexes most likely accounts for the efficiency of RNA degradation in vivo.

Chez les eucaryotes, la synthèse des ARN messagers (ARNm) est un processus hautement régulé en réponse à la fixation d’activateurs spécifiques sur des régions régulatrices. Cette étape permet le recrutement de co-activateurs, des facteurs généraux de la transcription (GTFs) et de l’ARN polymérase II (Pol II) pour former le complexe de préinitiation (PIC). Le Médiateur est un complexe co-activateur essentiel à ce processus et bien qu’il ait fait l’objet de nombreuses études ces dernières années, sa complexité a empêché de parvenir à une compréhension détaillée de son mécanisme de fonctionnement in vivo. Au cours de ma thèse, je me suis intéressée à la sous-unité Med17 qui joue un rôle central au sein du module de tête du Médiateur et interagit directement avec la Pol II. Nous avons construit une collection de mutants thermosensibles de cette sous-unité chez la levure Saccharomyces cerevisiae, que nous avons ensuite caractérisés par différentes approches de biologie moléculaire et génomique fonctionnelle. Nos analyses par ChIP-seq montrent que le Médiateur influence indépendamment le recrutement et/ou la stabilisation de la TBP ainsi que des modules cœur et kinase de TFIIH sur le génome. Ces résultats indiquent que, contrairement à la séquence d’assemblage linéaire observée in vitro, l’assemblage du PIC in vivo est un processus à plusieurs étapes non-séquentielles et que le Médiateur est important pour orchestrer l’arrivée des différents composants du PIC. Par ailleurs, nous avons mis en évidence un contact direct entre le Médiateur et Rad2/XPG, une endonucléase qui intervient dans la réparation de l’ADN. Une analyse à l’échelle du génome a révélé que cette protéine est présente sur les gènes de classe II, en absence de stress génotoxique et que sa localisation génomique corrèle avec celle du Médiateur. Nous avons ainsi démontré que le Médiateur est important pour le recrutement de Rad2, suggérant un nouveau rôle pour ce complexe dans la réparation de l’ADN, en plus de son rôle de co-activateur dans la transcription par la Pol II. / In eukaryotes, the synthesis of messenger RNA (mRNA) is highly regulated in response to the binding of specific activators to regulatory regions. This step allows the recruitment of coactivators, general transcription factors (GTFs) and RNA polymerase II (Pol II) to form the preinitiation complex (PIC). Mediator is a co-activator complex essential to this process and although it has been studied intensively during the last few years, its complexity has precluded a detailed understanding of the molecular mechanisms of its function in vivo. During my PhD, I focused on the Med17 subunit which plays a central role within the Mediator head module and interacts directly with Pol II. We obtained a large collection of temperature-sensitive mutants of this subunit in the yeast Saccharomyces cerevisiae, and then characterized these mutants by different molecular biology and functional genomics approaches. Our ChIP-seq analyses show that Mediator influences independently the recruitment and/or the stabilization of TBP as well as TFIIH core and kinase modules on the genome. These results indicate that, unlike a linear sequence observed in vitro, in vivo the PIC assembly is a non-sequential multistep process and that Mediator is important to orchestrate the recruitment of different PIC components. Furthermore, we identified a direct contact between Mediator and Rad2/XPG, an endonuclease involved in DNA repair. A genome-wide analysis reveals that this protein is present on class II genes in the absence of genotoxic stress, and that its genomic localization correlates with that of Mediator. We thus demonstrated that Mediator is important for Rad2 recruitment, suggesting a new role for this complex in DNA repair, in addition to its co-activator role in Pol II transcription.

No description available.

Abstract: Molecular and cellular insights into IKAP and Elongator functions As the first step in the complex process of gene expression, the transcription of genes from DNA to RNA by RNA polymerase II is subject to a multiplicity of controls and is thereby the endpoint of multiple cell regulatory pathways. We focused here on the molecular and cellular functions of IKAP and by extension of Elongator complex, initially found associated with the hyperphosphorylated RNA polymerase II during the elongation stage of transcription. IKAP is required for the assembly of Elongator subunits into a functional complex. Elongator has a histone acetyltransferase (HAT) activity associated with one of its subunits, named hELP3. In agreement with a potential role in transcript elongation, Elongator is associated with nascent RNA emanating from the elongating RNA polymerase II along the transcribed region of several yeast genes and chromatin immunoprecipitation experiments have also demonstrated an association of Elongator with genes in human cells. Different mutations in the human IKBKAP gene, encoding IKAP/hELP1, cause familial dysautonomia, a severe neurodevelopmental disease with complex clinical characteristics. Affected individuals are born with the disease and abnormally low numbers of neurons in peripheral nervous ganglions. To gain insight into the role played by IKAP and the Elongator complex in the transcription of genes and concomitantly learn about the molecular defects underlying the FD, an RNA interference approach was used to deplete the IKAP protein in human cells. In yeast, disruption of ELP1 (yeast homolog of human IKAP) is known to destabilize the ELP3 catalytic subunit, which leads to loss of Elongator integrity. Our experiments performed in human cells revealed that the levels of hELP3 protein is also affected by IKAP depletion after RNAi. We took advantage of this cellular loss-of-function model to identify genes whose transcription requires IKAP, by microarray experiments. Among the identified candidates, several were previously described to be involved in cell motility, or actin cytoskeleton remodelling. Because cell motility is of crucial importance for the developing nervous system, and therefore of obvious relevance to FD, the potential role of IKAP in cell motility was characterized at the cellular level. Several cell motility/migration assays demonstrated that the IKAP depletion has functional consequences so that IKAP-depleted cells showed defects in migration. Particularly, the reduced cell motility of neuronal-derived cell lines may be highly relevant to the neurodevelopmental disorder that affects FD patients. Whether or not the defects in cell migration resulted of impaired transcriptional elongation of the IKAP-dependent genes was investigated by chromatin immuno-precipitation technique. These experiments indicated that IKAP depletion leads to a decreased histone H3 acetylation in the transcribed region of its target genes in the context of Elongator complex. These acetylation defects are correlated with a decrease of the RNA polymerase II recruitment through the transcribed region of target genes, whereas the recruitment on the promoter is mostly unaffected. These results indicate that Elongator affects transcript elongation in vivo, but not the recruitment of the RNA polymerase II to the promoter. These very specific effects of IKAP/hELP1 depletion on histone acetylation and RNA polymerase II density across target genes are consistent with a direct effect of Elongator on transcriptional elongation in vivo and point to a function for Elongator in histone acetylation during transcript elongation. Résumé: Caractérisation des rôles biologiques de la protéine IKAP et du complexe Elongator La transcription des gènes de lADN en ARN est fondamentale pour lexpression des protéines et la capacité de nos cellules à sadapter à leur environnement. Ce processus finement régulé est catalysé par un enzyme, lARN polymérase II, vers lequel convergent une multitude de voies de signalisation. Dans le cadre de ce travail, nous nous sommes intéressés aux fonctions moléculaires et cellulaires de la protéine IKAP et du complexe Elongator. IKAP est la protéine qui assemble les sous unités dElongator en un complexe fonctionnel. Le complexe Elongator est associé à lARN polymérase II hyper-phosphorylée pendant létape délongation de la transcription et possède une activité histone acétyltransferase associée à une de ses sous unités, appelée ELP3. Chez la levure, Elongator est recruté an niveau des ARNs naissants, qui émanent directement de lARN polymérase II au niveau de la région transcrite des gènes étudiés. De plus, des expériences dimmunoprécipitation de la chromatine ont mis en évidence la présence du complexe Elongator au niveau de plusieurs gènes humains. Différentes mutations au niveau du gène IKBKAP, codant pour la protéine IKAP, sont responsables de la dysautonomie familiale, une maladie génétique qui affecte le développement du système nerveux périphérique. En effet, les individus affectés présentent une diminution de la densité de neurones au niveau des ganglions nerveux périphériques. Lobjectif de nos travaux est de comprendre davantage le rôle de la protéine IKAP et du complexe Elongator dans la transcription des gènes et ainsi, dinvestiguer les mécanismes moléculaires responsables dans la physiopathologie de la dysautonomie familiale. Un modèle de perte de fonction pour la protéine IKAP a dabord été généré par interférence dARN. Des travaux réalisés chez la levure indiquent que la protéine ELP1 (homologue de IKAP chez la levure) est essentielle pour la stabilité de la sous unité catalytique du complexe, la protéine ELP3. Les expériences réalisées sur notre modèle humain démontrent que le taux de la protéine ELP3 est également affecté par la déplétion dIKAP causée par linterférence dARN. Ce modèle de perte de fonction a été utilisé afin détablir la liste des gènes dont lexpression est contrôlée par la protéine IKAP, par des expériences de microarrays. Parmi les candidats identifiés, plusieurs ont été décrits comme impliqués dans la migration cellulaire et le remodelage du cytosquelette dactine. Le processus de migration des cellules est fondamental au cours du développement du système nerveux et par conséquent particulièrement relevant dans le contexte de la dysautonomie familiale. Limplication dIKAP dans la migration cellulaire a été investigué par différents tests de fonction qui montrent que la diminution dIKAP dans différentes lignées cellulaires entraîne une réduction significative de leur capacité migratoire. Ces résultats suggèrent que la diminution du nombre de neurones observée dans les ganglions périphériques des patients atteints de la dysautonomie familiale pourrait résulter dune altération de leur capacité à migrer au cours du développement. Enfin, des expériences dimmunoprécipitation de la chromatine ont été menées en utilisant notre modèle afin de déterminer dans quelle mesure le déficit de migration observé en labsence dIKAP serait la conséquence dun défaut de la fonction dElongator au niveau de lélongation de la transcription des gènes. Les résultats nous ont montré que la diminution dexpression dIKAP entraîne une réduction de lacétylation des histones H3 dans la région transcrite de ses gènes cibles. De plus, ce déficit dacétylation est directement corrélé avec un désengagement progressif de lARN polymérase II le long de la région transcrite de ces gènes. Par conséquent, ces résultats démontrent que le complexe Elongator affecte lélongation des transcrits in vivo, mais pas le recrutement de lARN polymérase II au niveau du promoteur. Ces effets très spécifiques de labsence dIKAP sur lacétylation des histones et lengagement de la polymérase II dans la transcription des gènes cibles montrent quElongator exerce un rôle direct au niveau de lélongation de la transcription de ces gènes. De plus, ces résultats suggèrent que la fonction dElongator serait dacétyler les histones au cours de lélongation transcriptionnelle in vivo.

RNA polymerase II (Pol II) is the enzyme responsible for the synthesis of all mRNAs in eukaryotic cells. As the central component of the eukaryotic transcription machinery, Pol II is the final target of regulatory pathways. While the role for different Pol II associated proteins, co-activators and general transcription factors (GTFs) in regulation of transcription in response to different stimuli is well studied, a similar role for some subunits of the core Pol II is only now being recognized. The studies reported in this thesis address the role of the fourth largest subunit of Pol II, Rpb4, in transcription and stress response using Saccharomyces cerevisiae as the model system. Rpb4 is closely associated with another smaller subunit, Rpb7 and forms a dissociable complex (Edwards et al., 1991). The rpb4 null mutant is viable but is unable to survive at extreme temperatures (>34ºC and <12ºC) (Woychik and Young, 1989). This mutant has also been shown to be defective in activated transcription and unable to respond properly in several stress conditions (Pillai et al., 2001; Sampath and Sadhale, 2005). In spite of wealth of available information, the exact role of Rpb4 remains poorly understood. In the present work, we have used genetic, molecular and biochemical approaches to understand the role of Rpb4 as described in four different parts below: i) Studies on Genetic and Functional Interactions of Rpb4 with SAGA/TFIID Complex to Confer Promoter- Specific Transcriptional Control To carry out transcription, Pol II has to depend on several general transcription factors, mediators, activators, and co-activators and chromatin remodeling complexes. In the present study, we tried to understand the genetic and functional relationship of Rpb4 with some of the components of transcription machinery, which will provide some insight into the role of Rpb4 during transcription. Our microarray analysis of rpb4∆ strain suggests that down regulated genes show significant overlap with genes regulated by the SAGA complex, a complex functionally related to TFIID and involved in regulation of the stress dependent genes. The analysis of combination of double deletion mutants of either the SAGA complex subunits or the TFIID complex with rpb4∆ showed that both these double mutants are extremely slow growing and show synthetic growth phenotype. Further studies, including microarray analysis of these double mutants and ChIP (chromatin immunoprecipitation) of Rpb4 and SAGA complex, suggested that Rpb4 functions together with SAGA complex to regulate the expression of stress dependent genes. ii) Study of Genome Wide Recruitment of Rpb4 and Evidence for its Role in Transcription Elongation Biochemical studies have shown that Rpb4 associates sub-stoichiometrically with the core RNA polymerase during log phase but whether recruitment of Rpb4 is promoter context dependent or occurs only at specific stage of transcription remains largely unknown. Having discovered that Rpb4 can recruit on both TFIID and SAGA dominated promoters, it was important to study the genome wide role of Rpb4. Using ChIP on chip experiments, we have carried out a systematic assessment of genome wide binding of Rpb4 as compared to the core Pol II subunit, Rpb3. Our analysis showed that Rpb4 is recruited on coding regions of most transcriptionally active genes similar to the core Pol II subunit Rpb3 albeit to a lesser extent. This extent of Rpb4 recruitment increased on the coding regions of long genes pointing towards a role of Rpb4 in transcription elongation of long genes. Further studies showing transcription defect of long and GC rich genes, 6-azauracil sensitivity and defective PUR5 gene expression in rpb4∆ mutant supported the in vivo evidence of the role of Rpb4 in transcription elongation. iii) Genome Wide Expression Profiling and RNA Polymerase II Recruitment in rpb4∆ Mutant in Non-Stress and Stress Conditions Structural studies have suggested a role of Rpb4/Rpb7 sub-complex in recruitment of different factors involved in transcription (Armache et al., 2003; Bushnell and Kornberg, 2003). Though only few studies have supported this aspect of Rpb4/Rpb7 sub-complex, more research needs to be directed to explore this role of Rpb4/Rpb7 sub-complex. To study if Rpb4 has any role in recruitment of Pol II under different growth conditions, we have studied genome wide recruitment of Pol II in the presence and absence of Rpb4 during growth in normal rich medium as well as under stress conditions like heat shock and stationary phase where Rpb4 is shown to be indispensable for survival. Our analysis showed that absence of Rpb4 results in overall reduced recruitment of Pol II in moderate condition but this reduction was more pronounced during heat shock condition. During stationary phase where overall recruitment of Pol II also goes down in wild type cells, absence of Rpb4 did not lead to further decrease in overall recruitment. Interestingly, increased expression levels of many genes in the absence of Rpb4 did not show concomitant increase in the recruitment of Pol II, suggesting that Rpb4 might regulate these genes at a post-transcriptional step. iv) Role of Rpb4 in Pseudohyphal Growth The budding yeast S. cerevisiae can initiate distinct developmental programs depending on the presence of various nutrients. In response to nitrogen starvation, diploid yeast undergoes a dimorphic transition to filamentous pseudohyphal growth, which is regulated through cAMP-PKA and MAP kinase pathways. Previous work from our group has shown that rpb4∆ strain shows predisposed pseudohyphal morphology (Pillai et al., 2003), but how Rpb4 regulates this differentiation program is yet to be established. In the present study, we found that disruption of Rpb4 leads to enhanced pseudohyphal growth, which is independent of nutritional status. We observed that the rpb4∆/ rpb4∆ cells exhibit pseudohyphae even in the absence of a functional MAP kinase and cAMP-PKA pathways. Genome wide expression profile showed that several downstream genes of RAM signaling pathway were down regulated in rpb4∆ cells. Our detailed genetic analysis further supported the hypothesis that down regulation of RAM pathway might be leading to the pseudohyphal morphogenesis in rpb4∆ cells.

En la levadura S. cerevisiae, un incremento de la osmolaridad extracelular activa la vía de Hog1, lo que produce una compleja respuesta adaptativa. Entre las respuestas adaptativas que Hog1 coordina, está un importante cambio en el partón de expresión génica. La tesis presentada se centra en la respuesta a nivel de regulación génica, y en ella se ponen de manifiesto nuevos mecanismos por los cuales Hog1 regula la transcripción para inducir genes necesarios para la adaptación celular en respuesta a estrés osmótico. Este trabajo demuestra que Hog1 controla la iniciación y la elongación de la transcripción, interacciona con la RNA polimerasa elongando, y es reclutado en toda la región codificante de los genes que se inducen por estrés osmótico a traves del 3'UTR. Asimismo, Hog1 recluta el complejo remodelador de cromatina RSC para promover un dramático cambio en el posicionamiento de nucleosomas, permitiendo una correcta inducción de la expresión génica. / In the yeast S.cerevisiae, an increase in extra cellular osmolarity activates the Hog1 Pathway, which produces a very complex adaptive response. Among these adaptive responses coordinated by Hog1, there is an important change in the gene expression pattern. The presented Thesis focuses on the response triggered at the genomic level, showing novel mechanisms by which Hog1 regulates transcription to efficiently and properly induce a subset of genes critical for the cellular adaptation to osmotic stress. This work demonstrates that Hog1 promotes and regulates transcription not only at the initiation level, as was previously described, but it also interacts with the RNA Polymerase while elongating, and travels along the coding regions of genes induced upon osmotic stress through recognition of the 3'UTR. Furthermore, Hog1 recruits a chromatin-remodeling complex known as RSC to promote a dramatic change in nucleosome positioning of target genes, allowing a proper induction of the transcription

In eukaryotes, the first step of interpreting the genetic information is the transcription of DNA into RNA. For protein-coding genes, such transcription is carried out by RNA polymerase II. A special domain of RNA polymerase II, called the C-terminal domain (CTD), functions as a master controller for the transcription process by providing a platform to recruit regulatory proteins to nascent mRNA (Chapter 1-2). The modifications and conformational states of the CTD, termed the 'CTD code', represent a critical regulatory checkpoint for transcription. The CTD, found only in eukaryotes, consists of 26--52 tandem heptapeptide repeats with the consensus sequence, Tyr₁Ser₂Pro₃Thr₄Ser₅Pro₆Ser₇. Phosphorylation of the serines and prolyl isomerization of the prolines represent two major regulatory mechanisms of the CTD. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Understanding how those modifying enzymes recognize and regulate the CTD is important for expanding our knowledge on the transcription regulation and deciphering the 'CTD code'. During my PhD study, I studied the function of CTD phosphatases and prolyl isomerase in the CTD regulation using Scp1, Ssu72 and Pin1 as model regulators. Scp1 and Ssu72 are both Ser5 phosphatases. However, Ssu72 is an essential protein and regulates the global transcription while Scp1 epigenetically silences the expression of specific neuronal genes. Pin1 is a highly conserved phosphorylation-specific prolyl isomerase that recognizes the phospho-Ser/Thr-Pro motif within the CTD as one of its primary substrates in vivo. Among these enzymes, Scp1 is the focal point of this dissertation, as it was studied from different angles, such as enzymatic mechanism (Chapter 3 describes the capture of phospho-aspartyl intermediate of Scp1 as a direct evidence for the proposed two-step mechanism), specific inhibition (Chapter 4 describes the identification and characterization of the first specific inhibitor of Scp1), and its non-active-site contact with the CTD (Chapter 5 describes the structural basis of this contact). These studies are of great importance towards understanding the molecular mechanism of the dephosphorylation process of the CTD by Scp1. / text

Regulation of transcription by Stress Activated Protein Kinases (SAPKs) is an essential aspect for adaptation to extracellular stimuli. In mammals, the activation of the p38 SAPK results in the regulation of gene expression through the direct phosphorylation of several transcription factors. However, how p38 SAPK regulates the proper gene expression program of adaptation to stress as well as the basic mechanisms used by the SAPK remains uncharacterized. The results displayed in this manuscript show that the p38 SAPK plays a central role in the regulation of gene expression upon stress, as up to 80% of the upregulated genes are p38 SAPK dependent. Moreover, we also observed that a specific set of genes were upregulated in response to each specific stimuli, and just a small set of genes were commonly up-regulated by several stresses, which involves mainly transcription factors. In addition, we observed that, to proper regulate gene transcription, the p38 SAPK is recruited to stress-induced promoters via its interaction with transcription factors. Additionally, p38 activity allows the recruitment of RNA polymerase II and the MAPKK MKK6 to stress-responsive promoters. The presence of active p38 SAPK at open reading frames also suggests the involvement of the SAPK in elongation. Altogether, the results showed in this manuscript establish the p38 SAPK as an essential regulator in the transcriptional response to stress, as well as define new roles for p38 in the regulation of transcription in response to stress. / La regulación de la transcripción por las Proteínas Quinasas activadas por Estrés (SAPKs) es un aspecto esencial para la adaptación a los estímulos extracelulares. En mamíferos, la activación de la SAPK p38 da lugar a la regulación de la expresión génica a través de la fosforilación de varios factores de transcripción. Sin embargo, cómo p38 SAPK regula el programa de expresión génica de adaptación al estrés así como los mecanismos utilizados por la SAPK permanece sin caracterizar. Los resultados presentados en este manuscrito muestran que p38 SAPK juega un rol central en la regulación de la expresión génica en respuesta a estrés, ya que hasta el 80% de los genes inducidos son dependientes de p38 SAPK. También observamos que en respuesta a cada tipo de estrés se induce un grupo de genes específicos, y sólo hay una pequeña respuesta de genes comunes a los diferentes tipos de estrés la cual engloba principalmente factores de transcripción. Además, hemos observado que para regular la transcripción, p38 se recluta a los promotores de respuesta a estrés a través de su interacción con factores de transcripción. Asimismo, la actividad de p38 permite el reclutamiento de la RNA Polimerasa II y de la MAPKK MKK6 a los promotores inducidos por estrés. La presencia de p38 activa en las regiones codificantes sugiere su participación durante la elongación. En conjunto, los resultados mostrados en este manuscrito establecen a p38 como un regulador esencial de la transcripción en respuesta a estrés, así como definen nuevas funciones de p38 en la regulación de la transcripción en respuesta a estrés.

Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 135-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;